Chronic psychosocial stress increases the risk for inflammation-related colon carcinogenesis in male mice.

Patients with inflammatory bowel diseases (IBDs) have a higher risk of developing colorectal cancer (CRC) than the general population. Furthermore, chronic psychosocial stress increases the likelihood of developing IBD and multiple types of malignant neoplasms, including CRC. Here, for the first time, we investigate the effects of chronic psychosocial stress in male mice on an artificially induced CRC, by employing the chronic subordinate colony (CSC) housing paradigm in combination with the reliable azoxymethane (AOM)/dextran sodium sulfate (DSS) CRC model. Colonoscopy revealed that CSC mice showed accelerated macroscopic suspect lesions. In addition, more CSC mice developed low-grade dysplasia (LGD) and/or high-grade dysplasia (HGD) in the colonic tissue compared to the single-housed control mice (SHC). CSC mice showed an increased number of Ki67+ and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling epithelial cells in colonic tissue. Colonic liver receptor homolog-1 (LRH-1), cyclooxygenase II (COXII), tumor necrosis factor, forkhead box P3 (FoxP3) mRNA as well as colonic ß-catenin, COXII, and LRH-1 protein expression were also increased in CSC compared with SHC mice. Although the number of CD4+ Th cells was increased, a tendency toward a decreased colonic interferon-γ (IFN-γ) mRNA expression was observed. Furthermore, despite an increased percentage of CD3+ cells and CD3+/FoxP3+ double-positive cells within mesenteric lymph node cells of CSC mice, IFN-γ secretion from these cells was unaffected. Altogether, our results suggest that chronic psychosocial stress increases the risk for AOM/DSS-induced and, thus, inflammation-related CRC. Finally, assessment of additional time points may test whether the shift from tumor-protective Th1 cell to regulatory T-cell immunity represents a consequence of increased carcinogenesis or a causal factor involved in its development.

T cell-dependent protective effects of CpG motifs of bacterial DNA in experimental colitis are mediated by CD11c+ dendritic cells.

BACKGROUND: Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanosine (CpG) sequence motifs constitute the immunostimulatory components of bacterial DNA which potently activate innate immunity. Administration of CpG-ODNs before the onset of experimental colitis prevents intestinal inflammation by induction of colitis-suppressing T cells. AIMS: To identify the interplay between innate and adaptive immune cells finally leading to protective CpG-ODN effects in intestinal inflammation. METHODS: Total splenic cells or purified selected cell types (CD4(+)CD62L(+) T cells alone or with B cells or dendritic cells (DCs)) from BALB/c mice were (co)-incubated in vitro with CpG-ODN for 5 days and CD4(+)CD62L(+) cells were injected intraperitoneally into C.B.-17 SCID (severe combined immunodeficiency) mice. Splenic CD4(+)CD62L(+) T cells were isolated from transgenic donor mice in which CD11c(+) DCs were depleted by diphtheria toxin administration during CpG-ODN treatment and injected into C57BL/6 Rag2(-/-) recipients. Intestinal inflammation was evaluated by histological scoring and cytokine secretion of mesenteric lymph node cells. RESULTS: CpG-ODN treatment of total splenic cells but not of purified CD4(+)CD62L(+) cells reduced the colitogenic potential of transferred T cells. While CpG-ODN stimulation of co-cultured CD4(+)CD62L(+) and B-cells did not alter the colitogenic potential of T cells, co-incubation of CpG-ODN-stimulated DCs and CD4(+)CD62L(+) cells reduced the colitogenic potential of the T cell population. Depletion of CD11c(+) DCs during CpG-ODN administration in vivo abolished the protective CpG-ODN effects. CONCLUSIONS: CpG-ODN-dependent protective effects in experimental colitis act indirectly on CD4(+)CD62L(+) T cells. While the involvement of B cells could be excluded, CD11c(+) DCs were identified as key mediators of CpG-ODN-induced protection in experimental colitis.

Loss of CD103+ intestinal dendritic cells during colonic inflammation.

AIM: To investigate possible differences in dendritic cells (DC) within intestinal tissue of mice before and after induction of colitis. METHODS: Mucosal DC derived from intestinal tissue, as well as from mesenteric lymph nodes and spleen, were analyzed by fluorescence activated cell sorting (FACS) analysis. Supernatants of these cells were analyzed for secretion of different pro- and anti-inflammatory cytokines. Immunohistochemistry and immunofluorescence were performed on cryosections of mucosal tissue derived from animals with colitis as well as from healthy mice. RESULTS: It was shown that DC derived from healthy intestinal lamina propria (LP) represented an immature phenotype as characterized by low-level expression of costimulatory cytokines. In contrast to DC from spleen and mesenteric lymph nodes (MLN) that secreted proinflammatory cytokines, LP-DC produced high levels of the anti-inflammatory cytokine IL-10. After induction of murine colitis in a CD4(+)CD62L(+) transfer model or in chronic dextran sulfate sodium-colitis, a marked increase of activated CD80(+) DC could be observed within the inflamed colonic tissue. Interestingly, in contrast to splenic DC, a significant population of DC within MLN and colonic LP expressed the mucosal integrin CD103 which was lost during colitis. CONCLUSION: The constitutive secretion of anti-inflammatory cytokines by immature DC within the intestinal LP might regulate the homeostatic balance between mucosal immunity and tolerance. CD103(+) DC could mediate this important function.

Calcitriol analog ZK191784 ameliorates acute and chronic dextran sodium sulfate-induced colitis by modulation of intestinal dendritic cell numbers and phenotype.

AIM: To investigate the effects of ZK1916784, a low calcemic analog of calcitriol on intestinal inflammation. METHODS: Acute and chronic colitis was induced by dextran sodium sulfate (DSS) according to standard procedures. Mice were treated intraperitoneally with ZK1916784 or placebo and colonic inflammation was evaluated. Cytokine production by mesenterial lymph node (MLN) cells was measured by ELISA. Immunohistochemistry was performed to detect intestinal dendritic cells (DCs) within the colonic tissue, and the effect of the calcitriol analog on DCs was investigated. RESULTS: Treatment with ZK191784 resulted in significant amelioration of disease with a reduced histological score in acute and chronic intestinal inflammation. In animals with acute DSS colitis, down-regulation of colonic inflammation was associated with a dramatic reduction in the secretion of the proinflammatory cytokine interferon (IFN)-gamma and a significant increase in intereleukin (IL)-10 by MLN cells. Similarly, in chronic colitis, IL-10 expression in colonic tissue increased 1.4-fold when mice were treated with ZK191784, whereas expression of the Th1-specific transcription factor T-beta decreased by 81.6%. Lower numbers of infiltrating activated CD11c+ DCs were found in the colon in ZK191784-treated mice with acute DSS colitis, and secretion of proinflammatory cytokines by primary mucosal DCs was inhibited in the presence of the calcitriol analog. CONCLUSION: The calcitriol analog ZK191784 demonstrated significant anti-inflammatory properties in experimental colitis that were at least partially mediated by the immunosuppressive effects of the derivate on mucosal DCs.

CpG motifs of bacterial DNA essentially contribute to the perpetuation of chronic intestinal inflammation.

BACKGROUND & AIMS: Recently, we demonstrated a proinflammatory effect of cytosin-guanosin dinucleotide (CpG)-oligodeoxynucleotide (ODN) treatment in established dextran sulphate sodium (DSS)-induced colitis. Here, we investigated whether DNA derived from luminal bacteria plays a role in the perpetuation of chronic intestinal inflammation. METHODS: Toll-like receptor (TLR9)-deficient and wild-type (wt) control mice were used for the induction of chronic DSS colitis. Moreover, mice with established chronic colitis using different experimental models were treated with adenoviral ODN (AV-ODN) known to block CpG effects. Colonic inflammation was scored and cytokine production was quantified both in colonic tissue and draining mesenteral lymph node cells (MLC). RESULTS: Eight weeks after induction of chronic DSS colitis in TLR9-deficient mice, intestinal inflammation was significantly lower (-68%), and proinflammatory cytokine production was drastically reduced. Treatment of wt mice with chronic DSS-induced colitis with AV-ODN resulted in a significant amelioration of disease with a reduced histologic score (-43%) and reduced cytokine production of MLC (interleukin [IL]-6: -68%; interferon [IFN]-gamma: -48%) and RNA expression of the T helper (Th)1-specific transcription factor T-bet (-62%) in colonic tissue. Qualitatively, the same results were obtained in the severe combined immunodeficiency disease (SCID) transfer model of colitis and in spontaneous colitis in IL-10-deficient mice. CONCLUSIONS: Bacterial DNA derived from luminal bacteria contributes significantly to the perpetuation of chronic intestinal inflammation. Inhibition of the immune-stimulating properties of bacterial DNA using AV-ODN may offer a novel and specific tool for the treatment of inflammatory bowel disease.

Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis.

Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day -2 to day +7. Chronic colitis was induced by transfer of CD4(+) CD62L(+) T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-gamma], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number of E. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-gamma, 32,477 +/- 6,377 versus 9,734 +/- 1,717 [P = 0.004]; IL-6, 231 +/- 35 versus 121 +/- 17 [P = 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 +/- 0.2 versus 1.9 +/- 0.3 [P = 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and resident E. coli into MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and resident E. coli into MLN.

OX40/OX40L interaction induces the expression of CXCR5 and contributes to chronic colitis induced by dextran sulfate sodium in mice.

Interactions between APC and T lymphocytes have been implicated as a major factor contributing to inflammatory bowel disease. To test whether OX40/OX40L interaction plays a role in chronic intestinal inflammation, we induced chronic colitis using dextran sulfate sodium and treated the mice with a murine fusion protein (OX40-IgG). Treatment resulted in a dose-dependent and significant reduction of intestinal inflammation (46%) as measured by a histologic score. IL-10 and IL-5 production from mesenteric lymph node cells increased 20-fold and 18-fold, respectively. In colonic tissue, IL-10 mRNA levels increased and the expression of T-bet was decreased to 30%. IL-10 neutralization partly inhibited the beneficial effects of OX40-IgG treatment. Surprisingly, despite the reduction of inflammation we found the number and size of colonic lymphoid follicles increased, with an accumulation of CD4(+) cells in the mantle area. In contrast, the number of CD4(+) cells infiltrating the mucosa was significantly reduced, as was their CXCR5 expression (24-fold). We conclude that OX40/OX40L interaction contributes to the perpetuation of chronic colitis partly by suppressing IL-10 production. Furthermore, our data suggest that the OX40/OX40L-induced CXCR5 expression on CD4(+) cells may be important for the inflammatory process by allowing migration to the germinal center for further differentiation of CD4(+) cells before they infiltrate the chronically inflamed mucosa.